Regulation of oxidative stress-induced calcium release by phosphatidylinositol 3-kinase and Bruton's tyrosine kinase in B cells

Hydrogen peroxide stimulates a tyrosine kinase-dependent calcium release from intracellular stores, which is assumed to be achieved through the activation of phospholipase Cγ2 (PLCγ2) via a tyrosine phosphorylation mechanism in B cells. Here we show that H(2)O(2) induces both tyrosine phosphorylation on PLCγ2 and the activation of phosphatidylinositol 3-kinase (PI3K) in B cells, and that the phosphatidylinositol 3-kinase inhibitor, Wortmannin, partially inhibited the H(2)O(2)-induced calcium release without affecting tyrosine phosphorylation on PLCγ2. Overexpression of human Bruton's tyrosine kinase (Btk), which was activated by H(2)O(2), almost completely overcame the inhibition of calcium release by Wortmannin. The reversal of Wortmannin's inhibition by enhancing Btk concentration seemed unique to the H(2)O(2)-mediated effect, because Btk failed to overcome the inhibition of Wortmannin on B cell receptor-triggered calcium mobilization. Immunoblot analysis revealed that Btk formed stable complexes with several tyrosine-phosphorylated proteins, including PLCγ2, only in Btk-overexpressed cells on H(2)O(2) stimulation. Together, our data are consistent with the notion that PIP3 and/or a high concentration of Btk target the activated PLCγ2 to its substrate site for maximal catalytic efficiency.